Numerous methods for the chemiluminescent detection of analytes such as DNA, RNA, proteins, antibodies, antigens, haptens, drugs, hormones, infectious agents and the like are known. Chemiluminescent detection can be performed by labeling analytes or molecules which specifically bind an analyte with a compound which can be made to undergo a chemiluminescent reaction (direct labeling). Chemiluminescent detection can be performed by labeling analytes or analyte-binding compounds with an enzyme or similar catalyst which catalyzes the chemiluminescent reaction of an added compound (enzyme labeling). The popularity of these modes of detection arises, in part, from the levels of sensitivity and wide range of measurement which is possible. Other favorable properties include safety, since no radioisotopes are required, versatility in the methods of labeling and choice of detection devices. In addition, in a commonly used format, probes are labeled with different haptens such as biotin, fluorescein and digoxigenin. These haptens, in turn, are then bound with their corresponding ligands or antibodies conjugated with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The label enzymes are detected with their respective chemiluminescent substrates.
It is frequently desirable to be able to detect and/or quantify more than one analyte at a time in a single test system. Savings in time, reagents and materials can thereby be realized and assay protocols can be simplified. In some instances information from multiple tests is required, for example, in certain medical diagnostic procedures, the results of two or more tests must be analyzed in combination in order to reach any conclusion. An exemplary technique requiring testing for multiple analytes is genetic fingerprinting of DNA samples for forensic, human identification or paternity determination tests by restriction fragment length polymorphism (RFLP) analysis of Southern blotted DNA. In this technique, blots require probing with several probes and often the limited amount of DNA necessitates the stripping and reprobing of the same blot multiple times (Adams, D. E., (1988), Crime Lab Digest 15:106-108; Noppinger, K., G. Duncan, D. Ferraro, S. Watson and J. Ban, (1992), BioTechniques. 13:572-575.). In Northern blot analysis, the expression of a specific gene is measured at the messenger RNA (mRNA) level and the signal is normalized by reprobing the blot for mRNAs such as .alpha.-tubulin or .beta.-actin that are normally invariant in the cell. In a Western blot of multiple protein antigens, the antibodies hybridized in one step are stripped and reprobed with another set of antibodies in an additional step to obtain data for another protein.
Stripping and reprobing may result in the loss of membrane bound target nucleic acids (Noppinger, K., G. Duncan, D. Ferraro, S. Watson and J. Ban, (1992), BioTechniques. 13:572-575.) and proteins (Krajewski, S., J. M. Zapata and J. C. Reed, (1996), Anal. Biochem. 236:221-228) thus reducing detection sensitivity in the second and subsequent probing steps. A method which allows the detection and differentiation of more than one analyte in a test system would avoid the aforementioned drawbacks. The present invention describes a chemiluminescent detection method that provides a solution to this problem by achieving the sequential detection of two different target analytes on a single blot and eliminating the stripping and reprobing step.
A method is disclosed in a PCT application WO97/24460 for multiple chemiluminescent reporter gene assays. These assays are performed in solutions to detect the presence or quantity of two or more enzymes expressed by a reporter gene in a transfected cell. Use of a peroxidase enzyme is not disclosed as it is not a commonly used reporter enzyme in transfection experiments.
A method for using two or more enzymatically triggerable dioxetanes to simultaneously produce light of different wavelengths is disclosed in U.S. Pat. No. 4,931,223. Light emission is triggered from two or more different enzyme labels. Since all of the light emitting reactions are proceeding simultaneously, means for optically distinguishing the various signals is required, thus increasing complexity. A further disadvantage of this approach is the difficulty of finding multiple different fluorophores whose emission spectra do not overlap to some degree. When this occurs, signal from one label will be partially detected in the wavelength region of the signal from another label. Decreased measurement accuracy and precision result.
U.S. Pat. No. 5,656,207 describes dual chemiluminescent assays of two different analytes in a solution wherein the analytes or their binding partners are directly labeled with chemiluminescent compounds. The two signals are generated simultaneously and are distinguished kinetically or spectroscopically. Enzyme labels are not involved and no mechanism for stopping or controlling either reaction is disclosed.
U.S. Pat. No. 5,672,475 also discloses dual luminescent binding assays using two different chemiluminescent direct labels in a solution. The two chemiluminescent signals, one from a luminol derivative and the other from an acridinium ester compound, are generated separately by a change of pH process conditions. Enzyme labels are not involved. A step of treating the solution with nitric acid is involved which would render the method unusable for detecting analytes on a blotting membrane.
A radioactive method of sequential detection of blotted DNA and proteins has been reported in the literature. Signal distinguishable probes labeled with .sup.32 p, .sup.35 S, and digoxigenin have been used by simultaneous hybridizations and for differential or sequential autoradiography (Au L. C., K. J. Chang, C. M. Shih and G. W. Teh, BioTechniques. 16:680-683 (1994)). However, here the signal differentiation was based on the intensity of signal rather than on the qualitative signal differences as in the present methods.
A method for the sequential chemiluminescent detection of multiple antigens on western blots with the enhanced luminol HRP chemiluminescent substrate at each step has been reported (Krajewski, S., J. M. Zapata and J. C. Reed. Anal. Biochem. 236:221-228 (1996)). While this method is able to sequentially detect multiple analytes on a blot, it is more operationally complex than the present methods. The antigen-antibody-HRP complex in each detection step is detected by chemiluminescence and then rendered unreactive by reacting with a chromogenic substrate which deposits a colored product on the band. Although this method has the ability to sequentially detect multiple antigens, it is more cumbersome and labor intensive than the present methods since the blot needs to be reprobed with primary and secondary antibodies for each detection step. Moreover, each step requires the application of two detection reagents to report the presence of one analyte.
Several substances are known which inhibit or destroy peroxidase activity. Among these are hydrogen peroxide at high concentrations, imidazole, phenylhydrazine, (W. Straus, J. Histochem. Cytochem., 28(7), 645-652 (1980)) fluoride and cyanide ions (P. Tulkens, R. Wattiaux, Experientia, 24(3), 219-223 (1968)). A listing of several inhibitors appears in a publication by Pierce Chemical Co., Rockford, Ill. (1994-95 catalog pp. T-315, 316). U.S. Pat. No. 4,810,630 describes the use of a nonionic surfactant to inhibit endogenous peroxidase activity of whole blood in immunoassays using horseradish peroxidase conjugates with calorimetric detection.